The DNA binding domain of GATA-1 contains a number of lysine residues that are modified by acetylation sumolation, ubiquitination and phosphorylation, and these modifications may be important in specifying GATA-1 function. We are investigating the role of these residues in GATA-1 activity and in factor recognition. In collaboration with the laboratory of Masyuki Yamamoto, we have shown that 3 lysine residues in the GATA-1 DNA binding domain are critical to the function of GATA-1, and appear to contribute by allowing GATA-1 to self-associate. These residues are among the lysines that are acetylated by CBP/ p300, but acetylation does not seem to be involved in generating the observed phenotype. Two of these residues are in the N-linker and one in the C-finger. The residues are not required for DNA binding. GATA-1 mutated in these residues is unable to rescue GATA-1.05 knockdown mice from embryonic lethality because it cannot support definite erythropoiesis. Mutation of these residues (K to A) decreases the ability of GATA-1 to self-associate. These mice show both positive and negative affects on GATA-1 target genes, while the levels of other targets remain unchanged. This is consistent with the idea that GATA-1 self-association is important for only a subset of the genes it controls. A new GATA-1 target gene, the transferrin receptor, was identified through this study. In collaboration with the Bougnres lab, we have identified a complex GATA site in the promoter of the p110 subunit of the P13 kinase gene that may be involved in regulating insulin resistance. The C genotype of a previously identified T/C polymorphism was found to correlate with increased sensitivity to insulin in two cohorts of obese non-diabetic children. This polymorphism creates a strong GATA binding site between two weaker sites in the p110 gene promoter. Lymphocytes from multiple cohorts of obese children homozygous for the C polymorphism have 1.5 fold higher p110 mRNA levels, and 1.7 fold higher p110 protein levels than cohort members with the T genotype. The levels of the p85, the other subunit of the PI3 kinase, are the same throughout the cohorts. These increases most likely occur through enhanced activation of the p110 subunit gene by GATA-3. The C promoter is more active than its T counterpart in transient assays in GATA-3 containing cells. The C promoter has a higher affinity than the T for GATA-2 and -3, both of which are involved in adipogenesis. While lymphoctes, which are not physiologically relevant to insulin resistance, were used in these studies, insulin responsive tissues could not be collected from this group of children. All other known SNPs in the vicinity of the PI3K gene (N=12) have been analyzed and do not contribute to this phenotype. The number of patients currently totals 2500 with analysis completed on 2000 of these. PU.1 is one of the proteins that interacts with the GATA-1 DNA binding domain and there is reciprocal inhibition between the two proteins. The PU.1 transactivation(TAD) and DNA binding domains are involved in the interaction and we have noted that the PU.1 TAD shares structural homology with the TAD of p53. In collaboration with the laboratory of Jim Omichinski we have shown that the p53 TAD also interacts in vitro and in vivo with the GATA-1 DNA binding domain. The linker and C-finger of GATA-1 are required for the interaction.The proteins reciprocally inhibit thetransactivation activity of one another in an erythroid precursor cell line, 6C2. GATA-1 may be required to prevent p53 induction during the nuclear condensation and enucleation that precedes erythrocyte formation or during the polyploidization of megakaryocytes.In collaboration with Masi Yamamoto we are testing mutants of GATA-1 in rescue assays in GATA1.05 cells and mice to determine the role of this interaction.